PHCOG J.
ORIGINAL ARTICLE
In vitro Effectiveness of Acacia concinna Extract against
Dermatomycotic Pathogens
Mansuang Wuthi-udomlert1* and Omboon Vallisuta2
1
Department of Microbiology, 2Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand.
Acacia concinna (Wild.) D.C. is an important medicinal plant in Thailand and throughout Asian countries. Its dried pods
are traditionally utilized as herbal medicine to treat many health symptoms e.g. laxative, cough, antidandruff and skin
diseases. This investigation was performed in order to demonstrate the antimicrobial potential of different A. concinna
extracts against the fungal causative agents of ringworm and opportunistic infections of immunocompromised
populations. Phytochemical study showed that the crude extract of A. concinna pod consisted of alkaloids, flavonoids,
saponin and tannin but none of antraquinone and cyanotic glycosides. The extracts e.g. ethanolic Soxhlet extract and
chloroform extract from Soxhlet extractor, and lyophilized extract and macerated ethanolic extract were tested using
diffusion method against 35 isolates of dermatophytes and each 20 isolates of Candida albicans, Cryptococcus
neoformans and Penicillium marneffei. The inhibitory effects were observed by considering their average inhibitory
zone diameter (IZD) compared to that of ketoconazole’s. From the averaged IZDs of all fungal isolates, the antifungal
effectiveness of lyophilized, chloroform, ethanolic (Soxhlet) and ethanolic (macerated) extract was at 18.38, 18.34, 16.67
and 14.06 mm, respectively.
Key words: Candida, Cryptococcus, dermatophytes, traditional medicine, lyophilized extract, soap pod
INTRODUCTION
Dermatophytes are a group of three keratinophilic fungal
genera, Trichophyton, Epidermophyton and Microsporum, that
cause daily found dermatomycotic symptoms called tinea
or ringworm on different parts of human body, as well as
animals’. This contagious mycopathogens may spread among
human or from animal or soil to human.[1] Although
dermatophytoses do not express any life threatening but
their clinical appearances are one of cosmetic problems,[2-3]
mental annoyance and can be regarded as everyday
troublesomeness. Most of modern topical drugs available
are not always affordable by people who required a long
term treatment to clear those disturbing features, these cost
a nation high budget for the effective treatment of the daily
found mycoses.
Address for correspondence:
Mansuang Wuthi-udomlert, Department of Microbiology,
Faculty of Pharmacy, Mahidol University,
447 Sri Ayuthaya Rd; Rajthevi, Bangkok 10400, Thailand.
Email: [email protected]
DOI: 10.5530/pj.2011.19.13
Pharmacognosy Journal | January 2011 | Vol 3 | Issue 19
Apart from these troubles, Candida albicans and Cryptococcus
neoformans are involved as frequently found mycoses in
tropical regions, alongside with acquisition of penicillosis
from Penicillium marneffei which disseminated in patients
infected by human immunodeficient virus.[4-7] The interesting
essentials from plant extracts that possess activities against
dermatophytes might express similar effects upon these
yeast-like fungi and dimorphic fungi.
Fortunately, many of those who faced these difficulties
reside in regions where herbs are utilized as traditional
medicines to which formularies and recipes have been
accustomed. For this reason, using of traditional herbal
medicines is an alternative mode in biodiversity rich
countries, with the support of Government and by
regulations, scientific researches in related area are increasing
accordingly. Therefore, the effectiveness of native medicinal
plants and their extracts by various methods of extraction
can be used as alternative manipulation to treat many
common infections.
Acacia concinna (Wild.) D.C. or soap pod, is naturally found
and being cultivated throughout South Asian countries.
The grind legume fruit powder is good to clean hair for its
superior cleansing quality to give lustrous hair, promoting
69
www.phcogj.com
ABSTRACT
www.phcogj.com
Mansuang and Omboon: In vitro Effectiveness of Acacia concinna Extract against Dermatomycotic Pathogens
hair growth and also reducing dandruff. Besides, other
medicinal activities of this pod fruit are for phlegm expellant,
laxative, cough syrup, appetizer, skin disease and fever
treatment. A. concinna is recorded on the use as traditional
medicines i.e. the root is to treat fever and intestinal disorder,
stem and leaves for laxative, stem bark to expel phlegm
and spoilage, flowers to treat ligamentous dysfunction.
Therefore, this plant is now being cultivated in large
quantities for commercial purpose in India and the Far
East because of its versatile properties.[8-10]
demonstrate scientific proof for antifungal activities. The
pods of A. concinna were macerated in ethanol, maceration
in water and further dried out by the use of lyophilizer and
the extraction by using Soxhlet apparatus. Therefore,
ethanolic extract, aqueous lyophilized extracts and the Soxhlet
ethanolic and chloroform extracts, respectively were obtained.
All were tested against 35 isolates of dermatophytes and
each of 20 isolates of Candida albicans, Cryptococcus neoformans
and Penicillium marneffei by the use of diffusion method.
However, various approaches on medicinal plant usages
render several varieties of essential ingredients from plant.
Because medicinal plants are used on the basis of traditional
knowledge in which conventional household consumption
is made from fresh plant naturally growth or kitchen garden
cultivation. Most of the utilizations are squeezing to obtain
juice, boiling with water, maceration in local made alcohol
or modifying into preserved part of plant, honey pill, tablet,
powder, sachet etc. To fulfill the immediate requirement
of traditional medicines and to preserve the over-supply
of fresh cultivation of seasonal products, simple expediency
to keep raw products for long term storage gives rise to
the community the benefit in managerial skill of local raw
products. Therefore, instead of freshly used, the specific
part of plant can be macerated in a short time and aqueous
supernatant is lyophilized, the dried material obtained can
be conveniently reconstituted to readily deliver the exact
quantitative amount. The activities of this lyophilized extract
are compared with those from the use of conventional
Soxhlet apparatus.
MATERIALS AND METHODS
Biological activity of medicinal plant extracts can be assessed
by various techniques, from qualitative diffusion in agar
plates, quantitative assay of dilution in broth or in agar,
and the commercial E Test which combines those two
methods’ principles. Research studies reported the congenial
results between qualitative diffusion test and quantitative
dilution test. Not only for unicellular fungi e.g. Candida,[11-16]
or Geotrichum,[17] but also for filamentous fungi e.g.
dermatophytes,[18] Aspergillus,[19-20] opportunistic hyphomycetes
i.e. species of Fusarium, Cladosporium, and ascomycetes i.e.
Chaetomium spp. as well.[21] However, these were evaluated
for modern drugs activities, different protocols and several
conditions were employed and presented in various
interesting aspects. These scientific evidences revealed the
relationship of disc diffusion test and those of quantitative
tests; therefore, the likewise protocol should be effective
in the evaluation for bioactivity of medicinal plant extracts
to manifest the required substantial properties which lead
to further modification of extracts into novelty herbal drugs.
This research is aimed to find out whether the medicinal
plants appeared in the records of Thai traditional medicines
70
Plant materials and plant extracts
Matured and dried fruits of Acacia concinna (Wild.) DC. was
purchased from local suppliers and identified according to
the voucher specimens deposited in Department of
Pharmacognosy, Faculty of Pharmacy, Mahidol University,
and the forest herbarium of the Royal Forest Department,
Bangkok, Thailand.
Ethanolic extract and chloroform extract: using Soxhlet
extractor
Dried ground powder of A. concinna was submitted to
sequential extraction with chloroform and methanol using
Soxhlet apparatus, the extract obtained was vacuum
evaporated.
Ethanolic extract: maceration
A. concinna powder was overnight macerated under aseptic
condition in 95% ethanol (1:4, w/v); after filtered through
sterile gauze, the filtrate was vacuum evaporated.
Aqueous lyophilized extract: using lyophilizer
All steps in the following procedures were aseptically done.
Clean, dried ground fruit of A. concinna was macerated in
sterile distilled water at 1:4 w/v for 24 h. The supernatant
after filtering was quantitatively collected and further
processed with lyophilizer (Leybold-Haerareus, Lyovac
GT-2, Germany). Aqueous lyophilized extract was kept in
bottle with tight closed lid at -20° C.
Antifungal activity test
The antimicrobial activity was determined by agar diffusion
assay described as the followings.
Microbial cultures
Clinical strains of 35 isolates of dermatophytes and each
of 20 isolates of Penicillium marneffei, Candida albicans and
Pharmacognosy Journal | January 2011 | Vol 3 | Issue 19
Mansuang and Omboon: In vitro Effectiveness of Acacia concinna Extract against Dermatomycotic Pathogens
Inoculum preparation
Active growth of dermatophytes isolates and P. marneffei
with the appropriate selected characteristics was suspended
in Sabouraud dextrose broth (SDB) (Pronadisa, S.A. Spain),
to give the fungal density equivalent to turbidimetric No. 1
McFarland. Culture of Candida albicans and Cryptococcus
neoformans were done likewise but to the density of No. 0.5
McFarland.
Extracts and reference drug preparation
A. concinna extracts were prepared as followings: ethanolic
extract and chloroform extract from Soxhlet extractor were
diluted 1:1 with their corresponding solvent; aqueous
lyophilized extract was treated in similar manner with sterile
distilled water. All test extracts were incorporated onto 6
mm in diameter sterile blank disc (Schleicher & Schuell,
Germany) at 20 µl/disc. Ketoconazole (Siemsgluss & Sohn,
Germany) was dissolved in methanol to give the net amount
of drug at 20 µg ketoconazole/disc. Test control discs were
ethanol and chloroform at 20 µl/disc each. Test discs were
prepared and used immediately after optimally dried out
in sterile condition.
C. neoformans plates at 37° C. Diameter of each inhibitory
zone was 3-time measured at different radial positions and
arithmetically averaged. The figures obtained were considered
as the activity of test materials. Culture control of each
microorganism was included in similar manner to deliver
the perfect timing in measuring the inhibitory zones.
RESULTS
Two samples of ethanolic extract were accessed from
evaporating macerated sample and from Soxhlet extractor
yielded different percentages of extracts. The first mentioned
extract gave nearly twice amount compared to the Soxhlet
extract whereas aqueous extract gave the highest yield among
all (Table 1).
Phytochemical Analysis
Phytochemical properties of A. concinna fruit were alkaloids,
saponin, tannin, flavonoids and cardiac glycosides but no
antraquinone and cyanotic glycosides (Table2).
However, using equal amount of suspension of each extract
incorporated onto each disc, the different amount of ground
material of A. concinna employed for one single disc could
be calculated (Table 3).
Table 1: Percentage yield of A. concinna extract from
different extraction methods.
Test methods
Extract
Suspension of all isolates of dermatophytes and P. marneffei,
20 µl each, was top layer cultivated onto total amount of
20 ml SDA plates whereas suspension of C. albicans and
C. neoformans was aseptically swabbed onto 20 ml SDA
plates. After the surfaces of all test plates were properly
dried, at least duplicates of prepared discs of each extract
and ketoconazole were laid onto the surface of inoculated
plates. Inoculated plate of dermatophytes and P. marneffei
were incubated at room temperature while C. albicans and
Ethanolic
14.18
Lyophilized
26.22
Chloroform
10.67
Ethanolic (Soxhlet)
8.73
% yield
Table 2: Phytochemical properties of A. concinna fruit
Plant
A.. concinna fruit
Alkaloids
Saponin
Tannin
Flavonoids
+
+
+
+
Table 3: Antifungal activity of A. concinna extracts as inhibitory zone diameter (IZD)
Inhibitory Zone Diameter (mean ± SD, mm)
Ethanolic
Extract
(0.14)*
Lyophilized
Extract
(0.08)*
Ethanolic
(Soxhlet) extract
(0.23)*
Chloroform
Extract
(0.19)*
Ketoconazole
20 µg/disc**
Dermatophytes (35)
14.88 ± 0.48
21.66 ± 0.49
18.50 ± 0.45
27.69 ± 0. 52
32.22 ± 0.92
P. marneffei (20)
18.71 ± 0.55
19.80 ± 0.66
23.31 ± 0.66
17.90 ± 0.85
35.25 ± 0.59
C. albicans(20)
10.56 ± 0.71
10.67 ± 0.83
10.75 ± 1.05
12.33 ± 0.67
26.60 ± 0.48
C. neoformans(20)
12.09 ± 1.07
21.39 ± 0.55
14.13 ± 0.43
15.42 ± 1.07
31.89 ± 0.41
Arithemetic mean
14.06
18.38
16.67
18.34
31.49
Organisms (n)
*mg/disc = calculated mg of raw grinding A. concinna **approx. eqv. to 1 mg of 2% topical drug
Pharmacognosy Journal | January 2011 | Vol 3 | Issue 19
71
www.phcogj.com
Cryptococcus neoformans were obtained from Department of
Microbiology, Faculty of Pharmacy, Mahidol University.
All pure isolates were kept as stock strain at -20° C on
Sabouraud dextrose agar (SDA) (Pronadisa, S.A. Spain).
Mansuang and Omboon: In vitro Effectiveness of Acacia concinna Extract against Dermatomycotic Pathogens
Discussion
Acknowledgments
Phytochemical ingredients are continuously employed as
natural essentials which are being promoted in many regions
to preserve and to remunerate local resources. The versatile
utilities of A. concinna has appeared in diverse traditional
records with different stresses in certain property e.g. the
use as laxative is accepted and being included as the
traditional use in the National List of Essential Drugs and
herbal crude drugs in Thai traditional household remedies
since 2004.[22]
The content of this manuscript is a part of project granted
by The National Research Council of Thailand (NRCT).
We would like to thank the NRCT for their permission to
publish this article.
www.phcogj.com
This study employed various extraction methods of A.
concinna fruit and demonstrated the activity of those extracts
by a selected test on the basis of return value to support
further traditional plant use in public health care. Hence,
agar-based method or diffusion test was the method of
choice for the fact that this test is simple, less time consuming
in performing the test, less labor intensive, low cost, readable
result at different times without subculture confirmation,
and being reproducible method.
Considering the IZDs of overall isolates, dermatophytes
and P. marneffei exhibited the respective highest susceptibility
to chloroform extract and ethanolic extract from Soxhlet
extractor, respectively. The result from lyophilized extract
gave the best result on C. neoformans. All extracts exerted
less than one half efficacy on C. albicans in comparison
with those of reference drugs, ketoconazole. Nevertheless,
these results exhibited the effectiveness of all A. concinna
extracts to be selected for further formulation.
On calculation of the raw material used for each disc i.e.
calculation of the incorporated extract on disc, extract yield
and the first weight of raw material used for extraction,
the positive inhibitory reaction revealed that aqueous
lyophilized extract was the most economically advantageous
(0.08 mg raw powder per disc) compared with other extracts.
The second was macerated ethanolic extract (0.14 mg raw
powder per disc). This implied the fact that the use of A.
concinna was so simple and able to verify the mode of
utilization of ancient medicine e.g. using herbs with water
and/or alcohol.
From various extraction methods, the use of Soxhlet
apparatus which is generally employed and basically familiar
in pharmacognosy area requires a lot of resources: chemicals,
instrument, time consumed and personal expertise. However,
while the antifungal activity of A. concinna extracts are
apparently demonstrated, it is of interest to investigate
whether those activities are attributed to any of phytochemical
components e.g. alkaloids, saponin, tannin or flavonoids.
72
References
1.
Rippon JW. Medical mycology. The pathogenic fungi and the pathogenic
actinomycetes, 3rd ed. WB Saunders, Philadelphia, 1988; Pp 134.
2.
Gupta AK, Albreski D, Del Rosso JQ, Konnikov N. The use of the new oral
antifungal agents, Itraconazole, Terbinafine, and Fluconazole, to treat
onychomycosis and other dermatomycoses. Current Problems in
Dermatology 2001; 13(4):213-246.
3.
Scher RK. Onychomycosis is more than a cosmetic problem. Br. J.
Dermatol. 1994; 130(Suppl. 43):15.
4.
Vithayasai P, Vithayasai V. Atlas of HIV infection, 2nd ed. Support The
Children Foundation. O.S. Printing.1994; Pp 1,8,41.
5.
Andrianopoulos A. Control of morphogenesis in the human fungal
pathogen Penicillium marneffei. Int. J Med Microbiol. 2002; 292; 331-347.
6.
Chayakulkeeree M, Perfect JR. Cryptococcosis. Infect Dis Clin North Am.
2006;20(3):507-44.
7.
Supparatpinyo K, Khamwan C, Baosoung V, Sirisanthana T, Nelson KE.
Disseminated Penicillium marneffei infection in Southeast Asia. The
Lancet 1994; 344(8915):110-113.
8.
Poonam K, Singh GS. Ethanobotanical study of medicinal plants used by
the Taungya community in Terai Arc Landscape, India. J Ethnopharmacol.
2009; 123:167-176.
9.
Natarajan V, Natarajan S. Antidermatophytic activity of Acacia concinna.
Global J of Pharmacol. 2009; 3 (1):06-07.
10. Singh S, Pandy P, Kumar S. Traditional knowledge on the medicinal
plants of Ayurveda. Central Institute of Medicinal and Aromatic
Plants(CIMAP), India, Lucknow, 2000; Pp 281.
11. Barry AL, Pfaller MA, Rennie RP, Fuchs PC, Brown SD. Precision and
accuracy of fluconazole susceptibility testing by broth microdilution, Etest,
and disk diffusion methods. Antimicrob. Agents Chemother. 2002;
46(6):1781-1784.
12. Matar MJ, Ostrosky-Zeichner L, Paetznick VL, Rodriguez JR, Chen E,
Rex JH. Correlation between E-Test, disk diffusion, and microdilution
methods for antifungal susceptibility testing of fluconazole and
voriconazole. Antimicrob. Agents Chemother. 2003; 47(5):1647-1651.
13. Odabasi Z, Paetznick V, Goldstein BP, Rex JH, Ostrosky-Zeichner L. Disk
Diffusion-based methods for determining Candida parapsilosis
susceptibility to anidulafungin. Antimicrob. Agents Chemother. 2003;
47(9):3018-3020.
14. Sims CR, Paetznick VL, Rodriguez JR, Chen E, Ostrosky-Zeichner L.
Correlation between microdilution, E-test, and disk diffusion methods for
antifungal susceptibility testing of posaconazole against Candida spp. J
Clin Microbiol. 2006; 44(6):2105-2108.
15. Kiraz N, Dag˘ I, Yamac M, Kiremitci A, Kas¸ifog˘lu N, Akgun Y. Antifungal
activity of caspofungin in combination with amphotericin B against
Candida glabrata: comparison of disk diffusion, Etest, and time-kill
methods. Antimicrob. Agents Chemother. 2009:53(2): 788-790.
16. Mandras N, Tullio V, Allizond V, Scalas D, Banche G, Roana J, et al. In
Vitro Activities of fluconazole and voriconazole against clinical isolates of
Candida spp. Determined by disk diffusion testing in Turin, Italy. Atimicrob.
Agents Chemother. 2009; 53(4):1657-1659.
17. Girmenia C, Pizzarelli G, D’Antonio D, Cristini F, Martino P. In Vitro
Susceptibility testing of Geotrichum capitatum: comparison of the E-Test,
disk diffusion, and sensititre colorimetric methods with the NCCLS
M27-A2 broth microdilution reference method. Antimicrob. Agents
Chemother. 2003; 47(12):3985-3988.
Pharmacognosy Journal | January 2011 | Vol 3 | Issue 19
Mansuang and Omboon: In vitro Effectiveness of Acacia concinna Extract against Dermatomycotic Pathogens
18. Ferna´ndez-Torres B, Carrillo-Mun˜oz A, Inza I, Guarro J. Effect of culture
medium on the disk diffusion method for determining antifungal
Susceptibilities of dermatophytes. Antimicrob. Agents Chemother. 2006;
50(6); 2222-2224.
19. Arikan S, Yurdakul P, Hascelik G. Comparison of two methods and three
end points in determination of in vitro activity of micafungin against
Aspergillus spp. Antimicrob. Agents Chemother. 2003; 47(8):2640-2643.
21. Pujol I, Guarro J, Llop C, Soler L, Ferna´ndez-Ballart J. Comparison
study of broth macrodilution and microdilution antifungal susceptibility
tests for the filamentous fungi. Antimicrob. Agents Chemother. 1996;
40:2106-2110.
22. Chokevivat V, Chuthaputti A. The role of Thai traditional medicine in
health promotion. 6GCHP Bangkok, Thailand; 2005.
www.phcogj.com
20. Lo´pez-Oviedo E, Aller AI, Martı´n C, Castro C, Ramirez M, Pema´n JM, et
al. Evaluation of disk diffusion method for determining posaconazole
susceptibility of filamentous fungi: comparison with CLSI broth microdilution
method. Antimicrob. Agents Chemother. 2006; 50(3):1108-1111.
Pharmacognosy Journal | January 2011 | Vol 3 | Issue 19
73